Process for preparing (1R,5S,6S)-2-[(6,7-dihydro-5H-pyrazolo [1,2-a][1,2,4]triazolium-6-yl)]thio-6-[(R)-1-hydroxyethyl]-1-methyl-carbapenem-3-carboxylate and starting materials thereof

ABSTRACT

Novel and improved simple process for preparing carbapenem compound, (1R,5S,6S)-2-[(6,7-dihydro-5H-pyrazolo [1,2-a][1,2,4]triazolium-6-yl)]thio-6-[(R)-1-hydroxyethyl]-1-methyl-carbapenem-3-carboxylate represented by the following formula: ##STR1## This compound may be prepared by using a mercapto reagent, 6,7-dihydro-6-mercapto-5H-pyrazolo-[1,2-a][1,2,4]triazolium derivative of the following formula: ##STR2## wherein X.sup.⊖ is an anion charge.

This application is division of Ser. No. 07/633,540, filed Dec. 28,1990.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a process for preparing(1R,5S,6S)-2-[(6,7-dihydro-5H-pyrazolo[1,2-a][1,2,4]triazolium-6-yl)]thio-6-[(R)-1-hydroxyethyl]-1-methyl-carbapenem-3-carboxylaterepresented by the following formula (I) ##STR3## or a pharmacologicallyacceptable salt thereof.

2. Description of the Prior Art

Since the discovery of an antibiotic substance "thienamycin" from thenature [U.S. Pat. No. 3,950,357; J. Am. Chem. Soc., 100, 313 (1987)],many researches have been carried out to develop carbapenem antibiotics.As a result of the extensive study, there has been developed imipenem(INN) which is a practically available antibacterial agent for clinicaluse.

Recently, Kumagai et al. proposed (1R,5S,6S)-2-[(6,7-dihydro-5H-pyrazolo[1,2-a][1,2,4]triazolium-6-yl)]thio-6-[(R)-1-hydroxyethyl]-1-methyl-carbapenem-3-carboxylateof formula (I) above as carbapenem antibiotic. See U.S. Pat. Nos.4,866,171; 4,925,836; and 4,925,935 (the entire specifications of thesepatents are incorporated herein by reference). This compound iscarba-2-penem-3-carboxylic acid compound in which a β-methyl group isintroduced at 1-position and6,7-dihydro-5H-pyrazolo[1,2-a][1,2,4]triazolium-6-yl thio group which isrepresented by the following ##STR4## is introduced at 2-position of thecarbapenem skelton. Owing to the presence of these specificsubstitutions, the compound of formula (I) has superior antibacterialactivity with excellent chemical and physicochemical stabilities inliving bodies, and is extremely stable against dehydropeptidase (DHP)known as a kidney enzyme.

The only synthesis for preparation of(1R,5S,6S)-2-[(6,7-dihydro-5H-pyrazolo[1,2-a][1,2,4]triazolium-6-yl)]thio-6-[(R)-1-hydroxyethyl]-1-methylcarbapenem-3-carboxylateof formula (I) heretofore known in Kumagai et al. is from an iminointermediate. That is, according to Kumagai et al., the comound offormula (I) can be prepared by reacting a compound represented byformula (II): ##STR5## wherein R¹ is a carboxyl protecting group, andR^(a) is an acyl group,

with a mercapto reagent represented by formula (VII): ##STR6## whereinR^(b) is an amino protecting group, to give a compound represented byformula (VIII): ##STR7## wherein R¹ and R^(b) have the same meanings asabove, and subjecting the compound of the formula (VIII) to removal ofthe protecting groups R¹ and R^(b) to give the carbapenem compound ofthe formula (IX) ##STR8## and then, reacting the resulting compound offormula (IX) with formimidic acid ester to give the carbapenem compoundof formula (I).

One presumed course of reaction in the formation of the compound offormula (I) by the reaction of the compound of formula (IX) with theformimidic acid ester derivative is that by the reaction of the compound(IX) with the formimidic acid ester derivative,(1R,5S,6S)-6-[(R)-1-hydroxyethyl]-1-methyl-2-[(1,2-di-iminomethyl)-4-pyrazolidinyl]thiocarbapenem-3-carboxylicacid of the following formula ##STR9## is formed as an intermediate, andthis imino intermediate is transitory compound and undergoes cyclizationreaction to form the compound of formula (I).

Finding a method for preparing the compound(1R,5S,6S)-2-[(6,7-dihydro-5H-pyrazolo[1,2-a][1,2,4]triazolium-6-yl)]thio-6-[(R)-1-hydroxyethyl]-1-methylcarbapenem-3-carboxylateof formula (I) through a method other than from the imino intermediatehas been an objective of the research of the present inventors and theirassignee since the discovery of the useful therapeutic indications ofthe compound (1R,5S,6S)-2-[(6,7-dihydro-5H-pyrazolo[1,2-a][1,2,4]triazolium-6-yl)]thio-6-[(R)-1-hydroxyethyl]-1-methyl-carbapenem-3-carboxylate.Despite the efforts of the researches of the assignee to find a way tomake (1R,5S,6S)-2-[(6,7-dihydro-5H-pyrazolo[1,2-a][1,2,4]triazolium-6-yl)]thio-6-[(R)-1-hydroxyethylethyl]-1-methyl-carbapenem-3-carboxylatewithout going through the transitory intermediate, efforts heretoforehave been unsuccessful. One apparent method for making(1R,5S,6S)-2-[(6,7-dihydro-5H-pyrazolo[1,2-a][1,2,4]triazolium-6-yl)]thio-6-[(R)-1-hydroxyethyl]-1-methylcarbapenem-3-carboxylateis disclosed in Merck European Patent Application 168,707 (1986), thatis discussed in detail in Kumagai et al. U.S. Pat. No. 4,866,171 atcolumn 1, lines 22-49. There is a picture representation of the compound6,7-dihydro-6-mercapto-5H-pyrazolo[1,2-a]-[1,2,4-triazolium halide whichis a key compound for the synthesis in the European application. Nowherein the European application is there any disclosure of the synthesis ofa 6,7-dihydro-6-mercapto-5H-pyrazolo[1,2-a][1,2,4]triazolium halide noris any method for the synthesis of a6,7-dihydro-6-mercapto-5H-pyrazolo[1,2-a]-[1,2,4]triazolium halideavailable within the skill of the art.

SUMMARY OF THE INVENTION

The present invention provides a novel and improved simple process forpreparing (1R,5S,6S)-2-[(6,7-dihydro-5H-pyrazolo[1,2-a][1,2,4]triazolium-6-yl)]thio-6-[(R)-1-hydroxyethyl]-1-methyl-carbapenem-3-carboxylateby using a key mercapto agent,6,7-dihydro-6-mercapto-5H-pyrazolo[1,2-a][1,2,4]triazolium derivative.

In accordance with the invention, there is provided a process forpreparing (1R,5S,6S)-2-[(6,7-dihydro-5H-pyrazolo[1,2-a][1,2,4]triazolium-6-yl)]thio-6-[(R)-1-hydroxyethyl]-1-methyl-carbapenem-3-carboxylateof the following formula ##STR10## or a pharmacologically acceptablesalt thereof; which comprises reacting a compound represented by thefollowing formula ##STR11## wherein R¹ is a carboxyl protecting group,and R^(a) is an acyl group,

with 6,7-dihydro-6-mercapto-5H-pyrazolo[1,2-a][1,2,4]triazoliumderivative represented by the following formula ##STR12## wherein X⊖ isan anion charge, to give a compound represented by the following formula##STR13## wherein R¹ and X⊖ have the same meanings as above, and then,subjecting the resulting compound of formula (IV) to removal of thecarboxyl protecting group to give the carbapenem compound represented byformula (I).

In accordance with a second aspect of the invention, there is provided6,7-dihydro-6-mercapto-5H-pyrazolo[1,2-a][1,2,4]triazolium derivativerepresented by the following formula ##STR14## wherein X⊖ is an anioncharge. This compound of formula (III) is a key mercapto reagent as astarting material for synthesis of the present invention.

For the above purpose of the invention, the compound of formula (III)may be prepared by reacting pyrazolidine-4-yl-disulfide represented bythe following formula ##STR15## with formimidic acid ester to give6,7-dihydro-5H-pyrazolo[1,2-a][1,2,4]triazolium-6-yl-disulfiderepresented by the following formula ##STR16## wherein X⊖ is is the samemeaning as above, and then, reducing the resulting compound of formula(V) to form the compound represented by the formula (III).

Therefore, in accordance with further aspect of the present invention,there are provided compounds represented by formula (V) and (VI),respectively.

Furthermore, the present invention provides a process for preparing thecompound of formula (III).

DETAILED DESCRIPTION OF THE INVENTION

The remarkable characteristics of the carbapenem compound according tothe present invention are that the substituent at 2-position of thecarbapenem skelton is a6,7-dihydro-5H-pyrazolo[1,2-a][1,2,4]triazolium-6-yl thio group and thatit has superior antibacterial activity and resistance to DHP.

In accordance with the present invention, the carbapenem compoundrepresented by formula (I) may be prepared by a process described below.

The carbapenem compound of formula (I) can be prepared by

(a) reacting pyrazolidine-4-yl-disulfide represented by the followingformula ##STR17## with formimidic acid ester to give a6,7-dihydro-5H-pyrazolo[1,2-a][1,2,4]triazolium-6-yl-disulfiderepresented by the following formula wherein X⊖ is an anion charge,

(b) reducing the resulting compound of formula (V) to form a6,7-dihydro-6-mercapto-5H-pyrazolo[1,2-a][1,2,4]triazolium derivativerepresented by the following formula ##STR18## wherein X⊖ has the samemeaning as above,

(c) reacting the resulting compound of formula (III) with a compoundrepresented by the following formula ##STR19## wherein R¹ is a carboxylprotecting group, and R^(a) in an acyl group,

to give a compound represented by the following formula ##STR20##wherein R¹ and X⊖ have the same meanings as above,

(d) then, subjecting the resulting compound of formula (IV) to removalof the carboxyl protecting group to give the carbapenem compoundrepresented by formula (I) ##STR21##

More specifically, the carbapenem compound represented by formula (I)may be prepared in such a manner as described in detail below.

In the specification of the present application, the term "lower"qualifying a group of a compound means that the group or compound soqualified has from 1 to 7, preferably from 1 to 4, carbon atoms.

The term "lower alkyl" referred to herein stands for a straight-chainedor branched-chain hydrocarbon group having preferably from 1 to 6 carbonatoms and may include, for example, methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert.-butyl, n-pentyl, isopentyl, n-hexyl,isohexyl or the like.

The term "carboxyl protecting group" referred to herein stands for anygroup capable of protecting the carboxyl group of the compound involvedwithout adversely affecting any other substituents and the reactionsthat follow and may include, for example, an ester residue such as alower alkyl ester residue including, for example, methyl ester, ethylester, n-propyl ester, isopropyl ester, n-, iso-, sec- or tert.-butylester, n-hexyl ester or the like; an aralkyl ester residue including,for example, benzyl ester, n-nitrobenzyl ester, o-nitrobenzyl ester,p-methoxybenzyl ester or the like; and a lower aliphatic acyloxymethylester residue including, for example, acetoxymethyl ester,propionyloxymethyl ester, n- or iso-butyryloxymethyl ester,pivaloyloxymethyl ester or the like.

The term "acyl group" referred to herein stands for, in a narrowersense, a moiety obtainable by removing the hydroxyl group from thecarboxyl group of an organic carboxylic acid as well as, in a broadersense, any acyl group derived from an organic sulfonic acid or anorganic phosphoric acid. Such an acyl group may include, for example, alower alkanoyl group such as acetyl, propionyl, butyryl or the like, a(halo)lower alkyl sulfonyl group such as methanesulfonyl,trifluoromethanesulfonyl or the like; a substituted or unsubstitutedarylsulfonyl group such as benzenesulfonyl, p-nitrobenzenesulfonyl,p-bromo-benzenesulfonyl, toluenesulfonyl,2,4,6-triisopropylbenzenesulfonyl or the like; and diphenylphosphoryl.

The term "amino protecting group" referred to herein stands for groupsusually employed in peptide chemistry, for example, aromatic acyl groupssuch as phthaloyl, benzoyl, benzoyl substituted by halogen, nitro or alower alkyl of 1 to 4 carbon atoms (e.g. chlorobenzoyl, p-nitrobenzoyl,p-tert-butylbenzoyl, toluoyl), naphthoyl; phenylacetyl; phenoxyacetyl;benzenesulfonyl; benzenesulfonyl substituted by a lower alkyl of 1 to 4carbon atoms (e.g. p-tert-butylbenzenesulfonyl, toluenesulfonyl); acylderived from aliphatic or halogenated aliphatic carboxylic acid such asacetyl, valeryl, caprylyl, n-decanoyl, acyloyl, pivaloyl, halogenoacetyl(e.g. monochloroacetyl, monobromoacetyl, dichloroacetyl,trichloroacetyl); camphorsulfonyl; methanesulfonyl; esterified carboxylgroups such as ethoxycarbonyl, tertbutyloxycarbonyl,isobornyloxycarbonyl, phenyloxycarbonyl, trichloroethoxycarbonyl,benzyloxycarbonyl, etc.; carbamoyl groups such as methylcarbamoyl,phenylcarbamoyl, naphthylcarbamoyl, etc.; and the correspondingthiocarbamoyl groups

The term "anion charge" referred to herein stands for a counterpartanion charge to a quaternary ammonium cation charge, and may include,for example, hydroxy anion; alkoxy anion such as methoxy anion, ethoxyanion; halogeno anion such as chloride anion, bromide anion, iodideanion, fluoride anion; and "acid anion residue" described below.

The term "acid anion residue" referred to herein may include acidicanion moiety obtainable by removing hydrogen atom from the organic acid,for example, a lower fatty acid such as acetic acid, propionic acid,butyric acid or trifluoroacetic acid; a substituted or unsubstitutedaryl acid such as benzoic acid, p-nitrobenzoic acid or the like; a(halo)lower alkylsulfonic acid such as methanesulfonic acid ortrifluoromethanesulfonic acid; a substituted or unsubstitutedarylsulfonic acid such as benzenesulfonic acid, p-nitrobenzenesulfonicacid, p-bromobenzenesulfonic acid, toluenesulfonic acid,2,4,6-triisopropylbenzenesulfonic acid or the like; substituted orunsubstituted arylphosphoric acid such as diphenylphosphoric acid; andinorganic acid such as nitrous acid, nitric acid, sulfuric acid,hydrochloric acid, hydrobromic acid, perchloric acid, fluorobromic acidor the like.

The mercapto reagent,6,7-dihydro-6-mercapto-5H-pyrazolo[1,2-a][1,2,4]triazolium derivativerepresented by formula (III) to be employed as a starting compound inthe process for manufacturing the carbapenem compound of formula (I) maybe prepared in accordance with the following Reaction Scheme A.##STR22## wherein R² and R³ are, independently each other, a hydrogenatom or an amino protecting group (R² and R³ are not both hydrogen); R⁴is a lower alkyl group; and Y is an acid anion residue.

The step (a) involves the reaction of the oxidation of4-mercaptopyrazolidine derivative of formula (X) to givepyrazolidine-4-yl-disulfide derivative of formula (XII).

The oxidizing reaction of 4-mercaptopyrazolidine derivative of formula(X) may be carried out in such a manner as known oxidizing reaction ofthiol compound in the organic chemical field. For example, the reactioncan be carried out in a solvent inert in the reaction such aschloroform, dichloromethane or the like, in the presence of an oxidizingreagent such as hydrogen peroxide, per-acid, copper (II) chloride,bromine, iodine or halo-succinimide. An air-oxidizing reaction (in thepresence of a base) may also be used for this purpose. Preferredreaction condition may be achieved by using iodine as the oxidizingreagent or the air-oxidizing in the presence of iron powder.

The step (b) involves the reaction of 4-substituted pyrazolidinederivative of formula (XI) with sulfur compound to givepyrazolidine-4-yl-disulfide derivative of formula (XII).

The acidic anion residue for symbol "Y" in the compound of formula (XI)is acidic moiety obtainable by removing hydrogen atom from the acidmentioned before, and preferably Y may be halogen atom such as chlorineor bromide; or sulfonic acid residue such as methanesulfonyloxy residue,toluenesulfonyloxy residue or trifluoromethanesulfonyloxy residue.

The sulfur compound to be used in this reaction may be sulfur powder,hydrogen sulfide, sodium sulfide or the like.

The step (c) is a step of elimination of the amino protecting groups R²and R³ from the compound of formula (XII) obtained by the step (a) or(b) above to give pyrazolidine-4-yl-disulfide of formula (VI).

The removal of the protecting groups R² and R³ may be made by a reactionknown per se for removing a protective group, such as solvolysis orhydrogenolysis. In a typical reaction, the compound represented byformula (XII) may be treated, for instance, in a mixture of solventssuch as tetrahydrofuran-water, tetrahydrofuranethanol-water,dioxane-water, dioxane-ethanol-water, n-butanol-water or the likecontaining morpholino-propane sulfonic acid-sodium hydroxide buffersolution (pH 7), a phosphate buffer solution (pH 7), dipotassiumphosphate, sodium bicarbonate or the like, using hydrogen under 1 to 4atmospheric pressures, in the presence of a catalyst for hydrogenationsuch as platinum oxide, palladium-activated carbon or palladiumhydroxide-activated carbon at temperatures ranging from approximately 0°C. to approximately 50° C. for approximately 0.25 to approximately 4hours.

As a result, the compound of formula (VI) may be obtained as di or tetraacid addition salt by treating with an acid, and may include thefollowing acid addition salt; pyrazolidine-4-yl-disulfide di or tetrahydrochloride salt, di or tetra hydrobromide salt, di or tetrahydroiodide salt, di or tetra trifluoroacetic acid salt, di or tetratrifluoromethanesulfonic acid salt, and di or tetra toluenesulfonic acidsalt.

The step (d) involves the reaction of pyrazolidine-4-yl-disulfide offormula (VI) with a formimidic acid ester derivative of formula (XIII)to give 6,7-dihydro[1,2-a][1,2,4]triazolium-6-yl-disulfide representedby formula (V).

The reaction may be carried out by reacting the compound of formula (VI)with the formimidic acid ester derivative such as ethyl formimidatehydrochloride, methyl formimidate hydrochloride or benzyl formimidatehydrochloride in an inert solvent such as water, alcohol,tetrahydrofuran or acetone. The pH of the reaction medium may beadjusted from about 6 to about 8 by adding a base such as sodiumhydrogen carbonate, potassium hydrogen carbonate, sodium carbonate,potassium carbonate, sodium hydroxide, potassium hydroxide, sodiumacetate, potassium acetate or the like.

The quantity of the formimidic acid ester derivative of formula (XIII)is not critical and may vary appropriately in a range generally fromapproximately 1 mole to approximately 20 moles, preferably from 4 molesto 12 moles, per mole of the compound (V). The reaction temperature isnot limited to a particular range and may vary from about -78° C. toabout room temperature, preferably from about -20° C. to about 10° C.The reaction may be finished under such conditions as described abovegenerally in approximately 5 minutes to approximately 1 hour.

6,7-Dihydro-5H-pyrazolo[1,2-a][1,2,4]triazolium-6-yl-disulfide offormula (V) may be obtained from the reaction mixture as crystallineform in this reaction.

The step (e) is a step by which6,7-dihydro-6-mercapto-5H-pyrazolo[1,2-a][1,2,4]triazolium derivative offormula (III) may be prepared by reducing the compound of formula (V)obtained by the step (d) above.

The reducing reaction may be conducted in accordance with known reactioncondition employed for cleavage of sulfur-sulfur bond of disulfidecompound, for example, reduction with trialkylphosphine such astrimethylphosphine, triethylphosphine, tributylphosphine;triarylphosphine such as triphenylphosphine; reduction with metal; orreduction with sodium borohydride, lithium aluminum hydride or lithiumtriethylborohydride. Particularly, the reduction with tributylphosphinemay be preferably employed. The reaction is preferably conducted in aninert solvent such as water; alcohol such as methanol, ethanol orisopropanol; ether such as ethyl ether, tetrahydrofuran or dioxane; orthe mixture solvent thereof in the presence of the reducing reagent.

The reaction temperature and time are not limited to a particular rangeand may vary according to the reducing reagent to be used. Usually, thereaction temperature may be in a range from about -20° C. to about 100°C., preferably from about room temperature to about 50° C., and thereaction time may be from about 10 minutes to about 10 hours. Afterreducing reaction,6,7-dihydro-6-mercapto-5H-pyrazolo[1,2-a][1,2,4]triazolium derivative offormula (III) may be obtained as a quaternary ammonium salt of formula(III) by using conventional manner such as distillation, extraction,lyophilization, etc.

6,7-Dihydro-6-mercapto-5H-pyrazolo[1,2-a][1,2,4]triazolium chloride,bromide, iodide, trifluoroacetate, methanesulfonate orp-toluenesulfonate can be obtained by the reaction above

The carbapenem compound of the present invention,(1R,5S,6S)-2-[(6,7-dihydro-5H-pyrazolo[1,2-a][1,2,4]triazolium-6-yl)]thio-6-[(R)-1-hydroxyethyl]-1-methyl-carbapenem-3-carboxylaterepresented by formula (I) may be prepared in accordance with thefollowing Reaction Scheme B. ##STR23## wherein R¹, R^(a) and X⊖ have thesame meanings as above.

The step (f) involves the reaction of a compound of formula (II) withmercapto reagent, that is,6,7-dihydro-6-mercapto-5H-pyrazolo[1,2-a][1,2,4]triazolium derivative offormula (III), to give a compound of formula (IV).

The reaction of the compound of formula (II) with the mercapto reagentof formula (III) may be carried out, for instance, by reacting thecompound of formula (II) with the mercapto reagent of formula (III) inan excess amount ranging from about an equimolar amount to approximately1.5 molar amount in an appropriate solvent such as tetrahydrofuran,dichloromethane, dioxane, dimethylformamide, dimethylsulfoxide;acetonitrile, hexamethylene phosphoramide or the like, preferably in thepresence of a base such as sodium hydrogen carbonate, potassiumcarbonate, triethylamine, diisopropylethyl amine or the like at atemperature range from approximately -40° C. to approximately 25° C. forapproximately 30 minutes to approximately 24 hours.

The reaction described above provides the carbapenem compoundrepresented by formula (IV) in which the carboxyl group at the3-position thereof is protected by the carboxyl protecting group R¹. Theremoval of the protecting group R¹ may be made by a reaction known perse for removing a protective group, such as solvolysis orhydrogenolysis. In a typical reaction, the compound represented byformula (IV) may be treated, for instance, in a mixture of solvents suchas tetrahydrofuran-water, tetrahydrofuran-ethanol-water, dioxane-water,dioxaneethanol-water, n-butanol-water or the like containingmorpholino-propane sulfonic acid-sodium hydroxide buffer solution (pH7), a phosphate buffer solution (pH 7), dipotassium phosphate, sodiumbicarbonate or the like, using hydrogen under 1 to 4 atmosphericpressures, in the presence of a catalyst for hydrogenation such asplatinum oxide, palladium-activated carbon or palladiumhydroxide-activated carbon at temperatures ranging from approximately 0°C. to approximately 50° C. for approximately 0.25 to approximately 4hours.

As a result,(1R,5S,6S)-2-[(6,7-dihydro-5H-pyrazolo[1,2-a][1,2,4]triazolium-6-yl)]thio-6-[(R)-1-hydroxyethyl]-1-methyl-carbapenem-3-carboxylaterepresented by formula (I) is produced.

The carbapenem compound of formula (I) is extremely stable againstdehydropeptidase (DHP) and has superior antibacterial activity.

The production of the carbapenem compounds of the formula (I) accordingto the present invention will be described more in detail by way ofworking examples.

In the following description, the following symbols are used to have theparticular meanings

Ac: acetyl group

Z: benzyloxycarbonyl group

PNB: p-nitrobenzyl group

EXAMPLE 1 ##STR24##

To a solution of 377 g of hydrazine monohydrate in 760 ml of ethanol wasadded dropwise 726 ml of ethylformate at 0° C. over 1 hour, and thereaction mixture was stirred for 30 minutes at the same temperature andfor 14 hours at room temperature. Then the reaction mixture was added to1011 ml of acetone over 30 minutes, and the resultant solution wasstirred for 30 minutes at room temperature. After the reaction, thesolvent was removed under reduced pressure to give 721 g (96 %) ofCompound (3) as a white solid.

NMR(CDCl₃) δ: 1.92(s,3H), 2.00(s,3H), 8.65(d,1H,J=9.9Hz),

EXAMPLE 2 ##STR25##

To a solution of 1 g of Compound (3) in 10 ml of methanol was added 2.12g of 28 % sodium methoxidemethanol solution at room temperature, and themixture was stirred for 1 hour, and then refluxed for 30 minutes. Then,1 ml of allyl bromide was added to the reaction mixture at roomtemperature and the mixture was stirred for 1 hour at the sametemperature, and then refluxed for 30 minutes. After the reaction, themixture was cooled to room temperature, and neutralized by adding formicacid. After the reaction solvent was removed under reduced pressure, theresulting residue was purified by column chromatography to give 933 mg(67 %) of Compound (4) as a colorless oil.

NMR(CDCl₃) δ: 1.86(s,3H), 2.13(s,3H), 4.08-4.24(m,2H), 5.17-5.37(m,2H),5.66-5.91(m,1H), 7.93-8.07(m,1H).

EXAMPLE 3 ##STR26##

3.7 g of Compound (4) was dissolved in 37 ml of formic acid, and thesolution was stirred for 6 hours at room temperature. After thereaction, the solvent was removed under reduced pressure and the residuewas purified by column chromatography to give 3.1 g (80 %) of Compound(5) as a pale yellowish oil.

NMR(CDCl₃) δ: 1.79(brs,1H), 4.1-4.3(m,2H), 5.2-5.5(m,2H), 5.6-6.0(m,1H),7.9-8.4(m,3H).

EXAMPLE 4 ##STR27##

To a solution of 3.1 g of Compound (5) in 15 ml of dichloromethane wasadded a solution of 2.2 g of sodium bromide monohydrate in 6 ml ofmethanol. To this mixture, 2.16 ml of bromine in 5 ml of dichloromethanewas added dropwise over 30 minutes under ice-cooling, and the resultingmixture was stirred for 30 minutes at room temperature. After thereaction, 6 ml of aqueous solution of 7.1 g of NaHCO₃ and 5 ml ofsaturated aqueous sodium sulfite were added to the reaction mixture andstirred for 10 minutes under ice-cooling. The mixture was extracted withethyl acetate and the organic layer was dried over Na₂ SO₄. After thesolvent was removed off under reduced pressure, the resulting residuewas purified by column chromatography to give 5.24 g (95%) of Compound(6) as a colorless oil.

NMR(CDCl₃) δ: 1.96(brs,1H), 3.4-4.5(m,5H), 8.0-8.4(m,2H).

EXAMPLE 5 ##STR28##

5.5 g of K₂ CO₃ was added to an ice-cooled solution of 5.2 g of Compound(6) in 26 ml of acetonitrile and the mixture was stirred for 1 hour atroom temperature. After the removal of the precipitate, the solvent wasremoved under reduced pressure. The residue was purified by columnchromatography to give 3.2 g (90 %) of Compound (7) as a pale yellowishoil.

NMR(CDCl₃) δ: 1.68(brs,1H), 3.8-4.6(m,5H), 8.50(s,1H).

EXAMPLE 6 ##STR29##

To a solution of 3.0 g of Compound (7) in 15 ml of acetonitrile wasadded 2.4 g of potassium thioacetate, and the mixture was stirred for 5hours at room temperature. After removal of the precipitate byfiltration, the solvent was removed under reduced pressure. Theresulting residue was purified by column chromatography to give 2.65 g(91 %) of Compound (8) as a pale yellowish oil.

NMR(CDCl₃) δ: 1.61(brs,1H), 2.37(m,3H), 3.4-4.2(m,5H), 8.42(s,1H).

EXAMPLE 7 ##STR30##

To a solution of 2.5 g of Compound (8) in a mixture of 10 ml ofdichloromethane:methanol (4:1) was added 2.9 g of 28 % sodiummethoxide-methanol solution under ice-cooling, and the mixture wasstirred for 5 minutes under the same temperature. Then, formic acid wasadded to the reaction mixture for neutralization and the precipitate wasremoved off by filtration. The solvent was removed, and the resultingresidue was purified by column chromatography to give 1.7 g (90%) ofCompound (8) as a pale yellowish oil.

NMR(CDCl₃) δ: 1.83(d,1H,J=5.3Hz), 2.8-3.0(m,2H), 3.2-3.4(m,2H),3.9-4.1(m,1H), 8.45(s,1H).

EXAMPLE 8 ##STR31##

1.7 g of Compound (9) was dissolved in a mixture solution of 5.1 ml ofmethanol:water (2:1). To this 2.6 g of KHCO₃ and 340 mg of Fe(II)Cl₂.6H₂O were added and the mixture was stirred overnight at room temperature.After removal of the precipitate, the solvent was removed under reducedpressure and the residue was purified by column chromatography to give1.5 g (90%) of Compound (10) as a colorless oil.

NMR(CDCl₃) δ: 4.0-4.3(m,2H), 5.2-5.5(m,2H), 5.6-5.9(m,1H),8.0-8.3(m,2H). EXAMPLE 9 ##STR32##

To a solution of 8.12 g of Compound (11) in 75 ml of methanol:water(2:1) mixture were added 10.2 g of KHCO₃ and 1.37 g of Fe(II)Cl₂.6H₂ O,and the mixture was stirred for 8 hours at room temperature. After thereaction, the precipitate was removed off by filtration, and the solventwas concentrated under reduced pressure. The residue was dissolved inchloroform and dried over sodium sulfate. The solvent was removed underreduced pressure to give 7.25 g (90%) of Compound (12) as a colorlessoil.

NMR(CDCl₃) δ: 3.10-4.00(m,8H), 4.55-4.75(m,2H), 8,42(s,4H)

EXAMPLE 10 ##STR33##

7.25 g of Compound (12) was dissolved in a mixture solution of 11.4 mlof conc. HCl and 103 ml of methanol and the mixture was stirred for 6hours at room temperature. After reaction, the precipitate was collectedby filtration, and dried in vacuo to yield 5.08 g (85%) of Compound (13)as a white solid.

NMR(CDCl₃) δ: 3.39(dd,4H,J=3.8Hz,13.0Hz), 3.59(dd, 4H,J=6.76Hz,13.0Hz),3.94-4.02(m,2H).

EXAMPLE 11 ##STR34##

To a solution of 11.4 g of Compound (14) in ml of dichloromethane wereadded 0.56 ml of triethylamine and 508 mg of iodine, and the reactionmixture was stirred for 10 minutes at room temperature. After thereaction, the mixture was washed with a sodium thiosulfite solution anda saturated sodium chloride solution, and then dried over sodiumsulfate. After removal of the solvent under reduced pressure, theresulting residue was purified by silica gel column chromatography togive 939 mg (85.2%) of Compound (15) as a pale yellowish solid.

NMR(CDCl₃) δ: 3.25(m,2H), 3.40(m,2H), 3.70(m,2H), 4.10(m,4H),5.16(s,8H), 7.30(s,20H).

EXAMPLE 12 ##STR35##

A mixture solution of 742 mg of Compound (15) in 3.1 g of hydrobromicacid-acetic acid solution (25%) was stirred for 4 hours at roomtemperature. After the reaction, the resulting precipitate was collectedby filtration, and washed with 10 ml of ethyl acetate. This precipitatewas treated with methanol to give 420 mg (79%) of Compound (16) as apale brownish solid.

NMR(D₂ O) δ: 3.50(4H,m), 3.53-3.85(6H,m).

EXAMPLE 13 ##STR36##

852.2 mg of KHCO₃ was added to an ice-cooled solution of 568 mg ofCompound (16) in 30 ml of water to adjust the pH of the solution to7.10. Then, 2.112 g of ethyl formimidate hydrochloride was added to thissolution and the reaction mixture was stirred for 10 minutes under thesame condition. After the pH of the reaction mixture was adjusted to 5.5with 1N-HCl, the mixture was washed with 50 ml of ethyl acetate. Theaqueous solution was concentrated to dryness under reduced pressure, and30 ml of methanol was added to the residue. After removal of theprecipitate by filtration, the solvent was removed and the resultingresidue was purified by SP-207 column chromatography and the eluent waslyophilized to give Compound (17).

This Compound (17) obtained by the above step was dissolved in 10 ml ofmethanol and treated with 1 ml of trifluoroacetic acid. The solvent wasremoved under reduced pressure to give 317.2 mg (58.3%) of Compound (18)as a white crystal.

NMR(D₂ O) δ: 4.80-5.00(4H,m), 5.00-5.16(6H,m), 9.05(4H,s).

EXAMPLE 14 ##STR37##

To a solution of Compound (18) in 6 ml of water-tetrahydrofuran (1:1)was added 0.082 ml of tri-n-butylphosphine under ice-cooling, and themixture was stirred for 1 hour at the same temperature. After thereaction, 10 ml of water was added to the reaction mixture and themixture was washed with dichloromethane and ethyl acetate. The aqueouslayer was lyophilized to give 102.2 mg (90.9%) of Compound (19) as awhite solid.

NMR(D₂ O) δ: 4.50-4.70(2H,m), 5.00-5.20(3H,m), 9.00(2H,s).

EXAMPLE 15 ##STR38##

To an ice-cooled solution of 85.5 mg of Compound (19) and 199.6 mg ofCompound (20) in 3 ml of anhydrous acetonitrile was added dropwise 0.06ml of diisopropylethylamine, and the mixture was stirred for 1 hourunder the same temperature. After the reaction, the solvent was removedunder reduced pressure and the resulting residue was dissolved in 20 mlof ethylacetate and then the solution was centifuged for 5 minutes at3,000 rpm. After centrifugation, the supernatant was decanted. Thecentrifugation and decantation was repeated three times and eachsupernatant was collected and concentrated under reduced pressure. Theresulting residue was dissolved in 20 ml of chloroform and the mixturewas centrifuged, and the supernatant was decanted off. Then, everyresidue obtained by the above centrifugation was collected and dissolvedin methanol. After removal of methanol under reduced pressure, 134.8 mg(67%) of Compound (21) was obtained as a pale yellowish oil.

NMR(CD₃ OD) δ: 1.32(d,6H,J=6.0Hz), 3.35(m,1H), 3.65(m,1H), 4.20(m,1H),4.42(m,1H), 4.60-4.90(m,2H), 5.1-5.3(m,3H), 5.36(ABq,2H,J=13.7Hz),7.67(d,2H,J=8.5Hz), 8.21(d,2H,J=8.5Hz), 9.07(s,1H), 9.08(s,1H).

EXAMPLE 16 ##STR39##

To a solution of 84.9 mg of Compound (21) in 2.5 ml of 0.1 M acetatebuffer and 2.5 ml of n-butanol was added 25 mg of 10% palladium-carbon,and the catalytic hydrogeneration was carried out at room temperaturefor 1 hour under a pressure of 4.0 atmospheres. After removal of thecatalyst, the catalyst was washed with 20 ml of water. The solvent wascombined and the pH of the solution was adjusted to 4.8-5.6 by adding1N-NaOH solution and washed with 20 ml of n-butanol. The solvent wasconcen trated to 5 ml under reduced pressure, and the resulting residuewas purified by SP-207 column chromatography and recrystallized fromethanol to give 30 mg (60%) of Compound (21) as a white crystal.

NMR(D₂ O) δ: 1.29(d,3H,J=7.3Hz), 1.33(d,3H,J=6.3Hz), 3.44(dq,1H,J=7.3,9.5Hz), 3.56(dd, 1H,J=2.9, 6.2Hz), 4.30(quintet,1H,J=6.2Hz),4.34(dd,1H,J=2.9, 9.5Hz), 4.75-4.84(m,2H), 5.08-5.17(m,2H),4.98-5.04(m,1H), 9.06(s,1H), 9.07(s,1H).

What we claim is:
 1. A6,7-dihydro-5H-pyrazolo[1,2-a][1,2,4]triazolium-6-yl-disulfiderepresented by the following formula ##STR40## wherein X⊖ is an anioncharge.
 2. A 6,7-dihydro-6-mercapto-5H-pyrazolo[1,2-][1,2,4]triazoliumderivative represented by the following formula ##STR41## wherein X⊖ isan anion charge.